Research in the Baneyx laboratory intersects engineering, biology and nanotechnology. How proteins fold into intricate three-dimensional shapes - or why they sometimes fail to do so - has profound implications in medicine and biotechnology. We study the genetics, regulation and structure-function relationship of molecular chaperones, a class of proteins that help other polypeptides reach a correct conformation. We exploit this fundamental knowledge to facilitate the production of bioactive recombinant proteins in the bacterium Escherichia coli, to build biosensors, and to explore the connection between protein (mis)folding and neurodegenerative diseases.

In the nanobiotechnology arena, we use combinatorial techniques to isolate and evolve short peptides binding to inorganic or synthetic materials. We bring to bear a variety of experimental and theoretical approaches to better understand the mechanisms of solid adhesion. We engineer solid-binding peptides within well-characterized protein "scaffolds" and use the resulting designer proteins to nucleate, organize and assemble nanostructured materials, with the aim of building systems and devices exhibiting superior mechanical or opto-electronic properties.

Colorized SEM image of Escherichia coli cells.E. coli is a well characterized organism suitable for the high-level expression of recombinant proteins.

.Protein Folding, Molecular Chaperones and Protein Expression

In order to display biological activity, newly synthesized proteins must fold into a precise three-dimensional conformation stabilized by hydrogen bonds, van der Waals interactions, hydrophobic interactions, salt bridges and disulfide bonds. Although all the information necessary for a protein to reach a proper structure is contained in its amino acid sequence, recombinant proteins of therapeutic or commercial interest are often unable to properly fold when expressed at high levels in bacteria or higher cells. The resulting misfolded proteins may either be degraded by cellular proteases or, more commonly, become sequestered into insoluble - and usually inactive - proteinaceous aggregates known as inclusion bodies. Protein misfolding and aggregation is also associated with a number of neurodegenerative and degenerative disorders including Parkinson's, Alzheimer's, Huntington's, Creutzfeld-Jakob and Gaucher's diseases.

Molecular chaperones are a class of proteins that help other polypeptides reach a proper conformation or cellular location. An improved understanding of the physiological role and mechanism of action of molecular chaperones, and of how they cooperate with each other in the management of protein folding/misfolding, has implications in the high-yield low-cost production of recombinant proteins, in uncovering the etiology of certain human diseases, and in developing therapeutic treatments for these disorders.

We study the function, biochemistry, regulation and practical applications of molecular chaperones in heterologous protein production. To read publications from our laboratory on this topic, clik here. To learn more about E. coli molecular chaperones, click on your favorite chaperone below or follow this link.

The molecular chaperone network in the cytoplasm of E. coli. Nascent proteins requiring the assistance of molecular chaperones to reach a proper conformation first encounter ribosome-bound trigger factor (TF) or the DnaK-DnaJ system (KJ). Both chaperones bind solvent-exposed stretches of hydrophobic amino acids in partially folded substrate proteins, shielding them for the solvent and from each other, and precluding their aggregation. Following undocking from TF or GrpE-mediated release from DnaK, folding intermediates may reach a native conformation, cycle back to DnaK-DnaJ or be transferred to the central chamber of GroEL for folding at infinite dilution upon GroES capping. In times of stress (e.g., heat, acid shock or oxidative conditions), a subset of cellular proteins unfolds and aggregates (red arrows). The small heat shock proteins IbpA/B bind partially folded species on their surface to serve as a reservoir of folding intermediate until stress abates and folding chaperones (DnaK-DnaJ and GroEL-GroES) become available for their refolding. IbpA/B also intercalate within large protein aggregates to facilitate their dissolution and the reactivation by the disaggregase ClpB and the DnaK-DnaJ system. The holding chaperone Hsp33 becomes important under oxidative stress conditions, when reactive oxygen species damage cellular proteins and cause them to misfold. Hsp31 stabilizes unfolding intermediates under times of intense cellular stress and following growth medium acidification. Recombinant proteins that miss an early interaction with TF or DnaK-DnaJ, that undergo multiple cycles of abortive interactions with folding chaperones, or that titrate them out, accumulate within inclusion bodies (green arrows).

.Cellular Stress and Sensor Development

In the wild, microorganisms are continuously exposed to stressful conditions including temperature upshifts and downshifts, abundance or dearth of nutrients, and changes in the physico-chemical composition of the growth environment. To survive these fluctuations, cells have evolved remarkable adaptive mechanisms which typically involve a transient increase in the synthesis of protective proteins (including molecular chaperones) that are well equiped to handle stress. We are interested in gaining a global understanding of stress responses in E. coli and their implications on cell physiology, recombinant protein expression, and genetic redundancy. On a more pragmatic front, we are exploiting stress responses to search for new antibacterial agents and to develop whole-cell sensors that provide a chromogenic or optical response in the presence of target analytes. Follow this link for relevant reviews and publications from our laboratory.

Emission wavelength-shifted mutants of firefly luciferase. Firefly (P. pyralis) luciferase produces yellow-green light upon oxidation of D-luciferin in the presence of ATP and molecular oxygen. Using error-prone PCR, we have isolated mutant enzymes producing orange and red light and are exploring their applications in sensor design and opto-electronics.

.Bionanofabrication

Nanotechnology can be broadly defined as the exploration and exploitation of unique phenomena occurring at the atomic, molecular and supra-molecular scales to create materials, devices and systems exhibiting novel properties and functions. This emerging field has the potential to radically alter the way we build composite materials, design electronic, optical and magnetic devices, perform catalysis, study biology, and diagnose and treat human diseases. Nanomaterials displaying useful properties as a result of their size, composition and/or architecture are already finding their way to the clinic (e.g., quantum dots, noble metal nanoparticle and nanoshells, paramagnetic nanoparticles and carbon nanotubes) and their penetration in other sectors is accelerating. Yet, most nanomaterials are difficult and expensive to synthetize and traditional approaches do not have the flexibility and versatility that is needed for the construction of the active nanostructures and systems of tomorrow.

Protein-aided nanofabrication. A designer protein was used to nucleate ≈2 nm diameter particles of cuprous oxide (dark spots) and the resulting protein shell-cuprous oxide core nanoparticles were assembled onto a DNA circle by taking advantage of the built-in DNA binding activity of the designer protein.

In nature, proteins are the ultimate conductors of material synthesis, orchestrating the nucleation, growth and assembly of a variety of soft and hard tissues with precise control of phase composition and architecture from the nano to the mesoscales. Although natural biomineralizing proteins only work on a limited number of compositions, it is now possible to use combinatorial techniques to select small peptides binding to solid phases of engineering interest. Using standard molecular biology techniques, these peptides can be fused within the framework of other proteins to generate "designer proteins" that combine the solid-binding (and sometimes solid-synthesizing) ability of the guest peptide with the original properties of the host scaffold (e.g., self-assembly, ligand binding, catalysis, light emission...) As part of the Genetically Engineered Materials Science & Engineering Center (an NSF MRSEC), and in collaboration with the Schwartz laboratory, we are building and characterizing designer proteins that will be suitable for the biofabrication of functional hybrid nanomaterials with programmed composition, phase, and topology. We also use eubacterial and archaebacterial S-layer proteins, which have the ability to self-assemble into nearly all space group symmetries, to template the growth of inorganic nanostructures into precise patterns. The resulting surfaces may be used for the construction of highly sensitive sensors and have applications in catalysis and opto-electronic devices construction. Follow this link for relevant reviews and publications from the Baneyx lab.

Templated surfaces. The surface layer (S-layer) protein of S. ureae assembles in a square crystalline structure that can be used as a resist to template the growth of inorganic materials (bottom). Images have been colorized.

Mujacic, M., M. Bader and F. Baneyx. 2004. "Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions". Mol. Microbiol.51:849-859.

Thomas, J.G. and F. Baneyx. 1996. "Influence of a global deregulation of the heat-shock response on the expression of aggregation-prone proteins in Escherichia coli". Ann. New York Acad. Sci.782:478-485.